Targeting Cathepsin B with p-coumaric acid rescues WWP1-dependent proteasomal degradation of NLRP3 in hyperuricemic nephropathy

BACKGROUND: Hyperuricemic nephropathy (HN) is a prevalent metabolic disorder characterized by tubulointerstitial inflammation and fibrosis, driven primarily by aberrant NLR family pyrin domain containing 3 (NLRP3) inflammasome activation. However, effective therapies targeting this pathogenic axis remain elusive. We aimed to identify a pharmacological intervention for HN and elucidate its mechanisms. METHODS: A targeted phenotypic screen of natural phenolic compounds was conducted against uric acid (UA)-induced inflammasome responses. Multi-omics profiling, molecular docking, and multi-dimensional biophysical assays-including surface plasmon resonance, cellular thermal shift assays, and circular dichroism-and site-directed mutagenesis were utilized to identify and characterize the direct molecular target. The underlying post-translational regulatory mechanism was investigated through co-immunoprecipitation and ubiquitination assays. In vivo efficacy and short-term tolerability were evaluated in a rat model of HN. Clinical relevance was assessed via renal biopsies and clinical sample analysis. RESULTS: We identified p-coumaric acid (p-CA) as a potent pharmacological candidate. Mechanistically, p-CA engages the Cathepsin B (CTSB) active-site/occluding-loop region, involving residues Cys26 and His110, and suppresses CTSB enzymatic activity. Notably, we uncovered a post-lysosomal regulatory model in which UA-induced lysosomal CTSB leakage is associated with a CTSB activity-associated reduction in WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) protein abundance/stability, thereby impairing WWP1-dependent K48-linked polyubiquitination and proteasomal clearance of NLRP3 and promoting inflammasome activation. p-CA-mediated CTSB inhibition preserves WWP1 protein abundance, restores NLRP3 proteasomal clearance, and attenuates downstream pyroptosis, inflammation, and fibrogenesis. CTSB knockdown attenuated the additional protective effect of p-CA, while pharmacological CTSB inhibition phenocopied p-CA, supporting CTSB pathway dependence. Furthermore, in vivo evaluations demonstrated that p-CA exerts urate-independent renoprotection without overt short-term systemic toxicities associated with synthetic CTSB inhibitors. Clinically, aberrant CTSB and NLRP3 upregulation correlated with impaired renal function in patients from the hyperuricemia-associated CKD cohort. CONCLUSIONS: Our findings support the CTSB-WWP1-NLRP3 regulatory axis as a previously unrecognized pathogenic mechanism of HN, revealing how lysosomal damage drives inflammasome hyperactivation by disrupting ubiquitin-dependent clearance. The precise molecular mechanism linking CTSB activity to WWP1 turnover remains to be fully defined. Targeting CTSB with p-CA provides a promising prophylactic or co-treatment candidate for HN warranting further translational evaluation.